Cellular (mono)layers form barriers between different compartments of the body. The transport of molecules across these cell layers is highly regulated. For example, the brain and cerebrospinal fluid (CSF) are separated from the blood by the blood-brain barrier and the blood-CSF barrier. These barriers facilitate the transport of selected molecules between the different compartments, whereas they prevent or severely limit the transport of others. Other cell layers such as gut epithelial cells serve similar functions. Knowledge of the cellular transport mechanisms at these barriers is not only important for understanding these fundamental biological processes but is also critical for the design of therapeutic agents that can pass through these barriers. Further, elucidation of the cellular processes that determine whether and how a drug candidate trafficks through the cell layers of a tumor is fundamental to the design of effective anti-cancer therapeutics. Microscopy provides a natural tool for the investigation of subcellular trafficking processes. However, classical microscopes are designed to image only one focal plane at a time i.e. to image processes that are largely confined to the two dimensions corresponding to the focal plane of the microscope. The processes at cellular barriers are inherently three dimensional in nature and therefore typically cannot be imaged in one focal plane. In addition, the available methods for changing focal planes are usually too slow to follow the highly dynamic and complex pathways of these transport phenomena. This accounts for the limited knowledge that is currently available regarding the transport processes in and across cellular layers. To overcome the problems associated with the imaging of dynamic trafficking processes in three dimensions, we have introduced multifocal plane microscopy (MUM). To date this technology has been extensively validated for imaging of dynamics up to a depth of around 2.5 micrometers. Unfortunately, this depth is insufficient to image the cellular processes that are of interest here. Therefore the current application is devoted to the development of extensions of the current MUM technology to enable the imaging of significantly larger depths that are required to image transport processes across deeper cellular (mono)layers. This includes the implementation of novel data analysis approaches for such MUM data. Quantum dot (QD)-labeled proteins, such as immunoglobulin G molecules, will be used throughout the experimental testing of the MUM configurations. The high photostability of QDs makes them very well suited for use in such analyses, which typically involve imaging over extended time periods. The Specific Aims are: Specific Aim 1: To develop multifocal microscopy approaches for the imaging of thick cellular samples. Specific Aim 2: To develop data analysis algorithms for analysis of MUM data from thick samples. Specific Aim 3: To elucidate single molecule trafficking pathways in deep cellular samples.